There is a lack in understanding about the role of characteristic mixtures of osmolytes in normal body function, and in many disease conditions, including diabetes mellitus and cancer. The long-term goal is to understand the normal and abnormal roles of these osmolytes in humans, and to design strategies for the use of osmolytes in the treatment and prevention of disease. This project's objective is to understand how non-specific (preferential) interactions contribute to the apparent necessity of osmolyte mixtures observed in vivo. It is hypothesized that the requirement for complex osmolyte cocktails is intrinsic to aqueous mixtures of proteins, nucleotide ligands, and osmolytes. The rationale is that this research provides the basis for understanding the role that osmolyte mixtures play in so many important normal and abnormal cellular processes. These include normal tissue function in kidney and brain, and the pathological impact of osmolytes in many diseases. The central hypothesis will be tested by pursuing Specific Aims that address the major reasons why complex osmolyte mixtures may be necessary: disparate impact of osmolytes on proteins and nucleotides, which leads to the necessity of the mixtures;and non-additive action of osmolytes, which leads to complex, non-linear behavior. The impact of osmolytes on protein-nucleotide systems will be quantified through high-throughput methods. Thermal melts and measurements of effective concentrations will be performed on microplates, and conventional approaches will be used to verify the results. The initial model proteins are adenylate kinase, dihydrofolate reductase, and carbonic anhydrase, in conjunction with small nucleotide ligands, including ATP, AMP, NADP, and others. Thirteen osmolytes that are common in humans and throughout nature will be used in binary mixtures. This project is innovative in its idea that pathological osmolyte mixtures are a contributing factor to many deadly diseases. We expect that knowledge of the effects of natural osmolyte cocktails on proteins and nucleotides will allow differentiating between deleterious and beneficial mixtures, and elucidate osmolytes'contributing role in many diseases. Such information would be very valuable in identifying targets for the manipulation of the osmolyte content of cells through medication, supplements, or diet. This project is significant, because it overcomes a major block in understanding cellular pathophysiology, namely the question of what new properties emerge when osmolytes are combined in their natural mixtures with proteins and metabolites, thus opening new opportunities of ameliorating disease. Additional benefits of this kind of research include an enhanced understanding of protein folding, of protein crystallization, and of drug formulation. PUBLIC HEALTH RELEVANCE: This project investigates mixtures of osmolytes (small molecules that naturally occur in humans), and how some of these cocktails can lead to beneficial or detrimental effects on biomolecules. It is expected that these results will facilitate the search for efficient chemoprevention agents against various cancers, and other diseases in which osmolytes play a role.